Saccharomyces cerevisiae yeast strain with functional expression of a GLUT transporter gene

ABSTRACT

The invention relates to a strain of the yeast  Saccharomyces cerevisiae  which, owing to deletion of the genomic sequences, no longer synthesizes hexose transporters and, as a consequence, can no longer grow on substrates with hexoses as the only carbon source, and whose ability of growing on a substrate with a hexose as the only carbon source is restored when it expresses a GLUT4 gene.

FOREIGN PRIORITY CLAIM

This application claims the priority under 35 U.S.C. §119 of GermanApplication No. 101 06 718.6 filed Feb. 14, 2001, which is herebyincorporated by reference herein in its entirety.

Most of the heterotrophic cells transport glucose into the interior ofthe cell via specific transporter proteins. In the various organisms,different mechanisms which mediate glucose transport have evolved, thatis to say proton symport systems, Na⁺ glucose cotransporters,binding-protein-dependent systems, phosphotransferase systems andsystems for facilitated diffusion. In the case of eukaryotes, a glucosetransporter family, encoded, in mammals, by the GLUT genes and inSaccharomyces cerevisiae by the HXT genes, mediates glucose uptake viafacilitated diffusion. These transporters belong to a larger sugartransport superfamily and are characterized by the presence of 12transmembrane helices and a plurality of conserved amino acid residuesand motifs.

Glucose transport in mammals was the focus of a number of studies sincethe knowledge of the processes is highly important in diseasesassociated with deficient glucose homeostasis, such as, for example,diabetes mellitus or Fanconi-Bickel syndrome. Eight glucose transporters(GLUT1- to GLUT5, GLUT8, GLUT9/SLC2A9, GLUT9 (GenBank® accession No.Y17803)) have so far been identified, all of which contribute to thefacilitated uptake of glucose. The key roles of these transportersinclude the uptake of glucose into a variety of tissues, their storagein the liver, their insulin-dependent uptake into the muscle cells andadipocytes, and glucose measurement by the pancreatic β-cells.

GLUT1 mediates the transport of glucose into the erythrocytes and acrossthe blood-brain barrier, but is also expressed in many other tissues,while GLUT4 is limited to insulin-dependent tissue, mainly to muscle andfatty tissue. In these insulin-dependent tissues, controlling thetargeting of GLUT4 transporters into intracellular compartments orplasma membrane compartments constitutes an important mechanism forregulating glucose uptake. In the presence of insulin, intracellularGLUT4 is redistributed to the plasma membrane in order to facilitate theuptake of glucose. GLUT1 is also expressed in these insulin-dependenttissues, and its distribution in the cell is also affected by insulin,but to a lesser extent. In addition, the relative efficacy with whichGLUT1 or GLUT4 catalyzes the transport of sugar is not only determinedby the extent to which each transporter is targeted to the cell surface,but also by their kinetic properties. The fact that different glucosetransporter isoforms are coexpressed, and that glucose is metabolizedrapidly, has made studies into the role and detailed properties of eachglucose transporter isoform in these insulin-dependent tissues acomplicated task. Heterologous expression systems such as Xenopusoocytes, tissue culture cells, insect cells and yeast cells have beenused to solve these problems. However, it emerged that these systemspresented difficulties: too weak an activity of the heterologouslyexpressed transporters, intrinsic glucose transporters in these systems,the intracellular retention of many of the transporters, or indeed theproduction of inactive transporters.

No organism is known as yet which, besides a heterologous and functionalGlut4 glucose transport protein, expresses no further hexose transportprotein, in particular intrinsic hexose transport protein. This leads toa series of disadvantages in the search for compounds which are capableof modifying the transport properties of the Glut4 protein. Suchcompounds would be of considerable interest as components ofpharmaceuticals since it is known that Glut4 plays an important role inlowering the glucose concentration in the blood together with insulinand other factors. An organism which expresses a functional Glut4transport protein would allow the search for compounds which directlyaffect the Glut4 transporter. The side effects of such compounds wouldbe less pronounced since no signal-factor-mediated side effects wouldoccur. Moreover, handling and providing the material would be greatlyfacilitated if a yeast strain were available. An object of the inventionis therefore to provide a yeast strain which expresses a functionalGlut4 protein.

The invention relates to a strain of the yeast Saccharomyces cerevisiaewhich can no longer grow on substrates with hexoses as the only carbonsource, and whose ability of growing on a substrate with a hexose as theonly carbon source is restored when it expresses a GLUT4 gene. Such astrain can be generated for example by mutating or deleting the relevantgenomic sequences. Hexoses are understood as meaning aldoses having 6carbon atoms, such as glucose, galactose or mannose, and ketoses having6 carbon atoms, such as fructose or sorbose.

The invention furthermore relates to a strain of the yeast Saccharomycescerevisiae as described above, this strain comprising a GLUT4 gene.

In the yeast Saccharomyces cerevisiae, 17 hexose transporters andadditionally three maltose transporters are known, which are capable oftransporting hexoses into the yeast if they are expressed stronglyenough. A strain is known in which all of the transporters which arecapable of taking up hexoses have been removed by deletion. This strainnow only comprises the two genes MPH2 and MPH3, which are homologous tomaltose transport proteins. The two genes MPH2 and MPH3 are repressedwhen glucose is present in the medium. The generation andcharacterization of this yeast strain is described in Wieczorke et al.,FEBS Lett. 464, 123-128 (1999). This strain is not capable of growing ona substrate with glucose as the only carbon source. Mutants can beselected from this strain which, on the basis of a suitable vector,functionally express Glut1 (strain hxt fgy1-1). If a plasmid vectorwhich carries a GLUT4 gene under the control of a yeast promoter istransformed into the yeast strain hxt fgy1-1, however, only a very smallamount of glucose is transported. The functional expression of Glut4requires further adaptations of this yeast strain to allow a significantglucose transport by means of Glut4. Such yeast strains which take upglucose into the cells by means of a single glucose transporter Glut4can be isolated on substrates with glucose as the only carbon source. Tothis end, a yeast strain hxt fgy1-1, which carries a Glut4 gene underthe functional control of a yeast promoter, is transformed. These yeastcells which have been transformed in this manner are plated onto amedium which comprises glucose as the only carbon source and areincubated thereon. After a few days incubation at, for example, 30° C.,the growth of individual colonies is observed. One of these colonies isisolated. If the yeast plasmid is removed from this colony, no growthtakes place on the medium with glucose as the only carbon source. If ayeast vector, which carries a GLUT4 gene under the functional control ofa yeast promoter, is retransformed into this strain, which now no longercomprises vector plasmid, then this strain regains the ability ofgrowing on a medium with glucose as the only carbon source. Thegeneration of a Saccharomyces cerevisiae strain which makes possible theuptake of glucose by means of a Glut4 transporter is described in detailin the examples. This strain expresses no yeast hexose transporters andis capable of taking up hexoses, in particular glucose, into the cell bymeans of a gene for a Glut4 transporter, for example a gene which hasbeen transformed into this strain. Yeast strains with thischaracteristic have been deposited under the number DSM 14035, DSM 14036or DSM 14037 at the Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH, (DSMZ) in Braunschweig in compliance with theprovisions of the Budapest treaty on the international recognition ofthe deposit of microorganisms for the purposes of patent procedure(Table 1).

In a preferred embodiment of the Saccharomyces cerevisiae strain of thepresent invention, a GLUT4 gene is expressed under the functionalcontrol of a yeast expression promoter. The skilled worker is familiarwith suitable yeast expression promoters. They are, for example, theSOD1 promoter (superoxide dismutase), ADH promoter (alcoholdehydrogenase), the promoter for the acid phosphatase gene, the HXT2promoter (glucose transporter 2), the HXT7 promoter (glucose transporter7), the GAL2 promoter (galactose transporter) and others. For thepurpose of expression, the construct, which consists of a yeastexpression promoter and a GLUT4 gene, is part of a yeast vector. Tocarry out the expression, this yeast vector may be present as aself-replicating particle, independently of the yeast genome, or else bestably integrated into the yeast genome. In principle, suitable yeastvectors are all polynucleotide sequences which are capable ofmultiplication in a yeast. Yeast vectors which can be used in particularare yeast plasmids or yeast artificial chromosomes. Yeast vectorscomprise, as a rule, an origin of replication (2μ., ars) for startingreplication, and a selection marker which usually consists of anauxotrophism marker or an antibiotic resistance gene. Yeast vectorswhich are known to the skilled worker are, for example, BM272, pCS19,pEMBCYe23, pFL26, pG6, pNN414, pTV3 or others. In principle, the GLUT4gene of any species can be expressed. A GLUT4 gene from humans, mice orrats is preferably expressed. The polynucleotide and amino acidsequences for Glut4 are accessible, for example, via the followingGenban® accessions: M20747 (cDNA; human), EMBL:D28561 (cDNA; rat),EMBL:M23382 (cDNA; mouse), Swissprot:P14672 (protein; human),Swissprot:P19357 (protein; rat) and Swissprot:P14142 (protein; mouse).The GLUT4 gene is especially preferably expressed by means of the vectorYEp4H7-HsGlut4 (SEQ ID No. 9). The GLUT4 gene of this vector is of humanorigin. The skilled worker is familiar with the generation of a yeastvector comprising a GLUT4 gene for expression in cells. The generationof such a vector is described in the examples. A yeast vector comprisinga gene for expression is transformed into the yeast for the gene to beexpressed. Methods which are suitable for this purpose are, for example,electroporation or the incubation of competent cells by vector DNA.Transformation is a technique with which the skilled worker is familiarand which serves to introduce foreign DNA, in particular plasmids orvectors, to microorganisms such as yeasts or bacteria. Detailedprotocols for the transformation of yeasts, yeast vectors, selection ofyeast mutants or the expression of proteins in yeasts are found in themanual “Methods in Yeast Genetics, 1997: A Cold Spring Harbor LaboratoryCourse Manual; Adams Alison (Edt.); Cold Spring Harbor Laboratory; ISBN:0-87969-508-0”, with which the skilled worker is familiar. The evidencethat the GLUT4 gene has been expressed in a yeast according to theinvention can be provided in particular by Northern blotting, Westernblotting, glucose uptake studies and glucose conversion studies or othermethods. Northern blotting involves applying isolated RNA of theorganism to be studied to a support such as, for example, nitrocelluloseand fixing it thereon, followed by incubation of this support, which nowcontains the RNA of the organism, with radiolabeled orfluorescence-labeled DNA of a GLUT4 polynucleotide sequence. Theexpression of GLUT4-mRNA in a yeast according to the invention isevidenced by the appearance of black bands. In comparison, no blackbands are detected with the RNA of a yeast which is otherwise identical,but has not been transformed with a GLUT4-containing expression vector.Western blotting involves the evidence of the expressed protein afterapplying a protein extract of the organism to be studied to a membranesupport such as nitrocellulose via antibodies. Antibodies for the Glut4protein can be obtained, for example, from Alpha DiagnosticInternational, Inc., 5415 Lost Lane, San Antonio, Tex. 78238 USA. Theassay systems required for detecting the bound antibody can also beobtained from this supplier. The expressed Glut4 protein is detected incomparison with a yeast strain which is otherwise identical, but doesnot comprise Glut4 protein. When carrying out glucose uptake studies,radiolabeled glucose is supplied as the only carbon source to the testorganism. In contrast with a control strain, which is otherwiseidentical but does not comprise Glut4 transporter, the yeast with Glut4as the only glucose transporter transports this radiolabeled glucoseinto the interior of the cell. Glucose conversion can be tested onnutrient media which comprise glucose as the only carbon source. Incontrast to the control, which is otherwise identical but does notcomprise Glut4 transporter, the yeast strain with a Glut4 transportprotein as the only glucose transporter is capable of growing in thenutrient medium with glucose as the only carbon source. The skilledworker is familiar with these methods which have just been mentioned.Detailed descriptions are found, for example, in “Current Protocols inMolecular Biology; Edited by: F. M. Ausubel, R. Brent, R. E. Kingston,D. M. Moore, J. G. Seidman, J. A. Smith, K. Struhl; published by: JohnWiley & Sons; 2000 (currently updated)”.

The invention preferably relates to a strain of the yeast Saccharomycescerevisiae wherein a Glut4 gene from humans, mice or rats is expressed.

The invention especially preferably relates to a strain of the yeastSaccharomyces cerevisiae wherein a polynucleotide sequence comprising acoding region of a human Glut4 gene is expressed.

In a further preferred embodiment, the invention relates to one or morestrains of the yeast Saccharomyces cerevisiae deposited, for example,under the Accession No. DSM 14038, DSM 14039 or DSM 14040 at theDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,Braunschweig. These strains are listed in table 1. This list containsthe information on the yeast strains used, into which the plasmids weretransformed, on the plasmids and on the growing conditions for theseyeasts.

The present invention also relates to the generation of a strain of theyeast Saccharomyces cerevisiae according to the invention, which isobtainable by the following process steps:

a) Providing a yeast which can no longer grow on substrates With hexosesas the only carbon source;

b) Transformation of the yeast of a) by a plasmid encompassing a GLUT4gene which is under the functional control of a promoter which can beexpressed in yeast;

c) Plating a strain which has been transformed in accordance with b)onto a medium comprising a hexose as the only carbon source;

d) Selection of a strain which has been plated in accordance with c) andwhich grows on this medium and supports the uptake of hexoses by meansof the Glut-4 gene.

The invention also relates to growing such a strain.

To provide a yeast in accordance with the invention, a strain of a yeastSaccharomyces cerevisiae which can no longer grow on substrates withhexoses as the only carbon source, but whose ability of growing on asubstrate with a hexose as the only carbon source is restored when itexpresses a Glut4 gene, is isolated in a first step. This can beeffected by mutating or deleting the relevant genomic sequences whichencode the hexose transporters. Providing the yeast furthermore requiresgrowing this yeast. Growing takes place by standard methods ofmicrobiology in suitable media. Suitable media for growing a yeast are,for example, complete media, in particular YPD medium (yeastextract/peptone/dextrose medium) or selective media and others. Theyeast cells are grown in these media, separated from the medium bycentrifugation after growing, and, for the purposes of the method,suspended in an aqueous medium comprising, inter alia, buffersubstances, salts or other additives, to give an aqueous suspension. Theskilled worker will find information on growing yeasts in “Methods inYeast Genetics, 1997: A Cold Spring Harbor Laboratory Course Manual;Adams Alison (Edt.); Cold Spring Harbor Laboratory; ISBN:0-87969-508-0”, which has already been mentioned above.

In a preferred embodiment of providing a yeast strain as describedabove, a GLUT4 gene under the functional control of a promoter which canbe expressed in yeast is used for the transformation. To carry out thetransformation, reference may be made to “Methods in Yeast Genetics”,which has already been mentioned above.

A GLUT4 gene which is especially preferably used for the transformationfor generating such a yeast strain is a GLUT4 gene from humans, mice orrats. Moreover, a GLUT4 gene which is especially preferably used for thetransformation is one which is present in a polynucleotide sequence asshown in SEQ ID No. 9 or 10. SEQ ID No. 9 discloses the polynucleotidesequence of the yeast vector Yep4H7-HsGLUT4. This vector comprises apolynucleotide sequence under the functional control of the HXT7promoter, which polynucleotide sequence encodes the amino acid sequenceof the human GLUT4 gene. SEQ ID No. 10 comprises the polynucleotidesequence of vector H2rg4g2. The yeast plasmid H2rg4g2 carries a GLUT4gene of rats under the functional control of an HXT2 promoter.Functional control of the GLUT4 gene by the promoter means that, bymeans of the promoter, an mRNA is transcribed which can be translatedinto a Glut4 protein. As regards the disclosures of the GLUT4 sequencesand the methods used, reference may be made to what has already beensaid above.

The invention furthermore relates to a method of identifying a compoundwhich increases or reduces the amount of a hexose transported by meansof a Glut4 protein, with the following process steps:

a) Providing a strain of the yeast Saccharomyces cerevisiae according tothe present invention which expresses a GLUT4 gene;

b) Determining the amount of a hexose taken up by it;

c) Providing a compound;

d) Contacting a strain of the yeast provided in accordance with a) witha compound provided in accordance with c);

e) Determining the amount of a hexose which is taken up into the yeaststrain after contacting in accordance with d);

f) Identifying a compound which increases or reduces the amount of ahexose transported by means of a Glut4 protein by comparing the amountof the hexose taken up into the strain before and after contacting inaccordance with d), which is determined in accordance with b) and e).

To provide a yeast-according to the invention, a strain of a yeastSaccharomyces cerevisiae, which can no longer grow on substrates withhexoses as the only carbon source, but whose ability of growing on asubstrate with a hexose as the only carbon source is restored when itexpresses a GLUT4 gene, is isolated in a first step. This yeast strainis transformed by means of a yeast vector comprising a GLUT4 gene underthe functional control of a yeast promoter. The generation of such ayeast strain is described in the examples. Providing the yeastfurthermore requires growing this yeast. Growing takes place by standardmethods of microbiology in suitable media. Suitable media for growing ayeast are, for example, complete media, in particular YPD medium (yeastextract/peptone/dextrose medium) or selective media and others. Theyeast cells are grown in these media, separated from the medium bycentrifugation after growing, and, for the purposes of the method,suspended in an aqueous medium comprising, inter alia, buffersubstances, salts or other additives, to give an aqueous suspension. Theskilled worker will find information on growing yeasts in “Methods inYeast Genetics, 1997: A Cold Spring Harbor Laboratory Course Manual;Adams Alison (Edt.); Cold Spring Harbor Laboratory; ISBN:0-87969-508-0”, which has already been mentioned above.

The amount of a hexose which is taken up by a yeast strain provided ashas just been described above can be determined by uptake studies withradiolabeled glucose. To this end, a specific amount of yeast cells, forexample an amount with a wet weight of 60 mg per ml, is suspended in,for example, 100 μl of a buffer and treated with a defined amount of¹⁴C- or ³H-labeled glucose as the only carbon source. The cells areincubated, and defined amounts of the cells are sampled at specifictimes. The amount of glucose taken up is determined with the aid of LSC(liquid scintillation counting). However, the amount of a hexose whichis taken up by a yeast strain provided as has just been described abovecan also be determined by growth tests on media with glucose as the onlycarbon source. To this end, the growth rate of the strain is determinedafter addition of the compound, for example by regularly measuring theoptical density of the culture at 600 nm, and this value is comparedwith the growth rate of a control strain (for example yeast wild-typestrain).

Providing a compound is done in particular by chemical synthesis, or byisolating chemicals from biological organisms. Chemical synthesis mayalso be automated. The compounds obtained by synthesis or isolation canbe dissolved in a suitable solvent. Suitable solvents are, inparticular, aqueous solutions comprising a particular amount of anorganic solvent such as, for example, DMSO (dimethyl sulfoxide).

Contacting a strain of the yeast with a compound for identifying acompound which increases or reduces the amount of a hexose transportedby means of a Glut4 protein is done in particular in automatedlaboratory systems provided for this purpose. Such systems can becomposed of specifically prepared chambers with recesses, of microtiterplates, Eppendorf tubes or laboratory glassware. Automated laboratorysystems are generally designed for high throughput rates. A process likewhat has just been described which is carried out with the aid of anautomated laboratory system is therefore also referred to as HTS (highthroughput screening).

After contacting the yeast with the compound, the amount of a hexose, inparticular glucose, which is transported by the yeast cell into theinterior of the cell under these conditions is determined. To this end,the same procedure may be used which has already been described fordetermining the glucose uptake for a strain which has not been contactedwith a compound.

The identification of a compound which increases or reduces the amountof a hexose transported by means of a Glut4 protein is carried out bycomparing the amount of the hexose taken up into the strain before andafter contacting it with the compound.

The invention furthermore relates to a pharmaceutical comprising acompound which has been identified and, if appropriate, furtherdeveloped by the method which has just been described using the Glut4gene, and to adjuvants for formulating the pharmaceutical for thetreatment of diabetes or adiposity. The further development of acompound which has been identified means that, firstly, the specificitywith regard to the target protein, in this case Glut4, is improved,secondly that the availability in the animal or human organism isincreased and thirdly that any existing undesired side effects arereduced. To this end, a series of methods is available to the skilledworker, including, for example but not by way of limitation, the use ofpharmacological animal models such as diabetic rats or ob/ob mice, theuse of biochemical in-vitro measurements, the use of virtual structuremodels of compounds and of the Glut4 protein. Adjuvants for theformulation of a pharmaceutical make possible the conditioning of theactive substance with the purpose of tailoring the application,distribution and development of action of the active ingredient to theapplication in question. Examples of such adjuvants are fillers,binders, disintegrants or glidants such as lactose, sucrose, mannitol,sorbitol, cellulose, starch, dicalcium phosphate, polyglycols,alginates, polyvinylpyrrolidone, carboxymethylcellulose, talc or silicondioxide.

Diabetes is evidenced by the excretion of glucose together with theurine combined with an abnormal increase in the blood glucose level(hyperglycemia) owing to a chronic metabolic condition due to lack ofinsulin or a reduced insulin effect. The lack of, or reduced, insulineffect leads to incomplete absorption and conversion of the glucosetaken up into the blood by the body cells. In fatty tissue,insulin-antagonistic hormones have the effect of increasing lipolysis,which entails an increase in the free fatty acid levels in the blood.

Adiposity (obesity) is the abnormal weight gain owing to an energyimbalance due to excessive intake of calories, which constitutes ahealth hazard.

The invention furthermore relates to the use of a compound which hasbeen identified, and, if appropriate, further developed by a methodusing the Glut4 protein for the preparation of a pharmaceutical for thetreatment of diabetes or adiposity. Pharmaceuticals are dosage forms ofpharmacologically active substances for the therapy of diseases orbodily malfunctions in humans and animals. For example, powders,granules, tablets, pills, lozenges, sugar-coated tablets, capsules,liquid extracts, tinctures and syrups are known for oral therapy.Examples which are used for external administration are aerosols,sprays, gels, ointments or powders. Injectable or infusible solutionsallow parenteral administration, with vials, bottles or prefilledsyringes being used. These and other pharmaceuticals are known to theskilled worker in the field of pharmaceutical technology.

The invention furthermore relates to a method of identifying a compoundwhich increases or reduces the amount of a hexose transported by meansof a Glut1 protein, with the following process steps:

a) Providing a strain of the yeast Saccharomyces cerevisiae which can nolonger grow on substrates with hexoses as the only carbon source andwhose ability of growing on a substrate with a hexose as the only carbonsource is restored when it expresses a GLUT1 gene, this straincomprising a GLUT1 gene under the functional control of a promoter whichcan be expressed in yeast;b) Determining the amount of a hexose which is taken up into this strainprovided in accordance with a);c) Providing a compound;d) Contacting a strain of the yeast provided in accordance with a) witha compound provided in accordance with c);e) Determining the amount of a hexose which is taken up into the yeaststrain after contacting in accordance with d);f) Identifying a compound which increases or reduces the amount of ahexose transported by means of a Glut1 protein by comparing the amountof the hexose taken up into the strain before and after contacting inaccordance with d), which is determined in accordance with b) and e).

To provide a yeast according to the invention, a strain of a yeastSaccharomyces cerevisiae which no longer forms hexose transporters owingto deletion of the genomic sequences and, as a consequence, is no longercapable of growing on substrates with hexoses as the only carbon source,but whose ability of growing on a substrate with a hexose as the onlycarbon source is restored when it expresses a GLUT1 gene, is isolated ina first step. Such strains are deposited at the Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH under the numbers DSM 14031, DSM14032 or DSM 14034.

In the yeast Saccharomyces cerevisiae, 17 hexose transporters andadditionally three maltose transporters are known, which are capable oftransporting hexoses into the yeast. A strain is known in which all ofthe transporters which are capable of taking up hexoses have beenremoved by deletion. The generation and characterization of this yeaststrain is described in Wieczorke et al., FEBS Lett. 464, 123-128 (1999).This strain is not capable of growing on a substrate with a hexose asthe only carbon source. If a plasmid vector which carries a Glut1 geneunder the control of a yeast promoter is transformed into such a yeaststrain, no glucose transport takes place nevertheless. The functionalexpression of Glut1 requires further adaptations of this yeast strain inorder to make possible the transport of glucose by means of Glut1. Suchyeast strains which take up glucose into the cells by means of a singleglucose transporter Glut1 can be isolated on substrates with glucose asthe only carbon source. To this end, a yeast strain which no longerexpresses intact hexose-transporting proteins is transformed with ayeast vector which carries a GLUT1 gene under the functional control ofa yeast promoter. These yeast cells which have been transformed in thismanner are plated onto a medium comprising glucose as the only carbonsource and are incubated thereon. After a few days incubation at, forexample, 30° C., the growth of individual colonies is observed. One ofthese colonies is isolated. If the yeast plasmid is removed from thiscolony, no growth takes place on the medium with glucose as the onlycarbon source. If a yeast vector carrying a GLUT1 gene under thefunctional control of a yeast promoter is now transformed into thisstrain, which no longer comprises vector plasmid, then this strainregains the ability of growing on a medium with glucose as the onlycarbon source.

To transform a yeast strain, a GLUT1 gene from humans, mice or rats isused in particular. Polynucleotide sequences and amino acid sequencesfor Glut1 are disclosed under the following code numbers of thedatabases indicated: EMBL:M20653 (cDNA; human), EMBL:M13979 (cDNA; rat),EMBL:M23384 (cDNA; mouse), Swissprot:P11166 (protein; human),Swissprot:P11167 (protein; rat), Swissprot:P17809 (protein; mouse).

The generation of such a yeast strain is described in the examples.Providing the yeast furthermore requires growing this yeast. Growingtakes place by standard methods of microbiology in suitable media.Suitable media for growing a yeast are, for example, complete media, inparticular YPD medium (yeast extract/peptone/dextrose medium) orselective media. The yeast cells are grown in these media, separatedfrom the medium by centrifugation after growing, and, for the purposesof the method, suspended in an aqueous medium comprising, inter alia,buffer substances, salts or other additives, to give an aqueoussuspension. The skilled worker will find information on growing yeastsin “Methods in Yeast Genetics, 1997: A Cold Spring Harbor LaboratoryCourse Manual; Adams Alison (Edt.); Cold Spring Harbor Laboratory; ISBN:0-87969-508-0”, which has already been mentioned above.

In a preferred embodiment of this method, a strain of the yeastSaccharomyces cerevisiae is provided which comprises a GLUT1 gene underthe functional control of a promoter which can be expressed in yeast.Such strains which are suitable for this method were deposited at theSammlung für Mikroorganismen und Zellkulturen GmbH under the number DSM14033, DSM 14026 or DSM 14033.

A GLUT1 gene as constituent of a plasmid is disclosed in SEQ ID No. 11or SEQ ID No. 12. SEQ ID No. 11 comprises the sequence of the yeastvector Yep4H7-HsGlut1. This plasmid comprises the polynucleotidesequence of a human GLUT1 gene under the functional control of an HXT7promoter. SEQ ID No. 12 comprises the polynucleotide sequence of theyeast vector H2rg1g2. This plasmid comprises the polynucleotide sequenceof a GLUT1 gene from rats under the functional control of the HXT2promoter.

The amount of a hexose which is taken up by a yeast strain provided ashas just been described above can be determined by uptake studies withradiolabeled glucose. To this end, a specific amount of yeast cells, forexample an amount with a wet weight of 60 mg, is suspended in, forexample, 100 μl of a buffer and treated with a defined amount of ¹⁴C- or³H-labeled glucose as the only carbon source. The cells are incubated,and defined amounts of the cells are sampled at specific times. Theamount of glucose taken up is determined with the aid of LSC (liquidscintillation counting).

Providing a compound is done in particular by chemical synthesis, or byisolating chemicals from biological organisms. Chemical synthesis mayalso be automated. The compounds obtained by synthesis or isolation canbe dissolved in a suitable solvent. Suitable solvents are, inparticular, aqueous solutions comprising a particular amount of anorganic solvent such as, for example, DMSO (dimethyl sulfoxide).

Contacting a strain of the yeast with a compound for identifying acompound which increases or reduces the amount of a hexose transportedby means of a Glut1 protein is done in particular in automatedlaboratory systems provided for this purpose. Such systems can becomposed of specifically prepared chambers with recesses, of microtiterplates, of Eppendorf tubes or of laboratory glassware. Automatedlaboratory systems are generally designed for high throughput rates. Aprocess like what has just been described which is carried out with theaid of an automated laboratory system is therefore also referred to asHTS (high throughput screening).

After contacting the yeast with the compound, the amount of a hexose, inparticular glucose, which is transported by the yeast cell into theinterior of the cell under these conditions is determined. To this end,the same procedure may be used which has already been described fordetermining the glucose uptake for a strain which has not been contactedwith a compound.

The identification of a compound which increases or reduces the amountof a hexose transported by means of a Glut1 protein is carried out bycomparing the amount of the hexose taken up into the strain before andafter contacting it with the compound.

The invention furthermore relates to a pharmaceutical comprising acompound which has been identified and, if appropriate, furtherdeveloped by the process which has just been described using the Glut1gene, and to adjuvants for formulating the pharmaceutical for thetreatment of diabetes or adiposity. The further development of acompound which has been identified means that, firstly, the specificitywith regard to the target protein, in this case Glut4, is improved,secondly that the availability in the animal or human organism isincreased and thirdly that any existing undesired side effects arereduced. To this end, a series of methods is available to the skilledworker, including, for example but not by way of limitation, the use ofpharmacological animal models such as diabetic rats or ob/ob mice, theuse of biochemical in-vitro measurements, the use of virtual structuremodels of compounds and of the Glut1 protein. Adjuvants for theformulation of a pharmaceutical make possible the conditioning of theactive substance with the purpose of tailoring the application,distribution and development of action of the active ingredient to theapplication in question. Examples of such adjuvants are fillers,binders, disintegrants or glidants such as lactose, sucrose, mannitol,sorbitol, cellulose, starch, dicalcium phosphate, polyglycols,alginates, polyvinylpyrrolidone, carboxymethylcellulose, talc or silicondioxide.

Diabetes is evidenced by the excretion of glucose together with theurine combined with an abnormal increase in the blood glucose level(hyperglycemia) owing to a chronic metabolic condition due to lack ofinsulin or a reduced insulin effect. The lack of, or reduced, insulineffect leads to incomplete absorption and conversion of the glucosetaken up into the blood by the body cells. In fatty tissue,insulin-antagonistic hormones have the effect of increasing lipolysis,which entails an increase in the free fatty acid levels in the blood.Adiposity (obesity) is the abnormal weight gain owing to an energyimbalance due to excessive intake of calories, which constitutes ahealth hazard.

The invention furthermore relates to the use of a compound which hasbeen identified and, if appropriate, further developed by a method usingthe Glut1 protein for the preparation of a pharmaceutical for thetreatment of diabetes or adiposity. Pharmaceuticals are dosage forms ofpharmacologically active substances for the therapy of diseases orbodily malfunctions in humans and animals. For example, powders,granules, tablets, pills, lozenges, sugar-coated tablets, capsules,liquid extracts, tinctures and syrups are known for oral therapy.Examples which are used for external administration are aerosols,sprays, gels, ointments or powders. Injectable or infusible solutionsallow parenteral administration, with vials, bottles or prefilledsyringes being used. These and other pharmaceuticals are known to theskilled worker in the field of pharmaceutical technology.

The invention furthermore relates to the polynucleotide sequence of SEQID No. 13 and to the polynucleotide sequence of SEQ ID No. 14. Thepolynucleotide sequences of SEQ ID No. 13 and 14 encode mutations of therat Glut1 gene which lead to the substitution of individual amino acidsin the protein in question. The polynucleotide sequence of SEQ ID No. 13encodes a Glut1 protein with a substitution of valine with methionine inposition 69 of the amino acid chain. The polynucleotide sequence of SEQID No. 14 encodes a Glut1 protein where an alanine is substituted withmethionine in position 70 of the amino acid chain. Both protein mutantssupport the uptake of glucose even in a strain whose hexose transportershave been switched off by deletion, but which does not yet support theuptake of glucose by the wild-type Glut1 protein. Such mutants can beobtained for example via selection for suppressor mutations or viain-vitro mutagenesis.

The invention furthermore relates to a Glut1 protein which is encoded bythe polynucleotide sequence of SEQ ID No. 13 or 14.

The invention also relates to yeast strains comprising a polynucleotidesequence of SEQ ID No. 13 or a polynucleotide sequence of SEQ ID No. 14.Such yeast strains are deposited at the Deutsche Sammlung fürMikroorganismen und Zellkuturen GmbH as DSM 14026 and DSM 14027. Togenerate these strains, yeast vectors corresponding to SEQ ID No. 13 or14 are transformed into a yeast strain which is no longer capable ofgrowth on substrates with hexoses as the only carbon source and whoseability of growing on a substrate with hexose as the only carbon sourceis eventually restored when a Glut1 gene is expressed in this strain.Then, after transformation, the cells are plated onto a medium withglucose as the only carbon source. The colonies growing on this mediumare isolated. The yeast strain which has been transformed in this manneris suitable for example for carrying out a method of identifying acompound which increases or reduces the amount of a hexose transportedby means of a Glut1 protein.

Abbreviations

HXT hexose transporter

ORF open reading frame

PCR polymerase chain reaction

EXAMPLES Growing the Yeast Strains

All of the yeast strains described herein were derived from strainCEN.PK2-1C (MATa leu2-3, 112 ura3-52 trp1-289 his3-Δ1 MAL2-8^(c) SUC2).The generation of a yeast strain with deletions in the hexosetransporter genes (HXT) was described by Wieczorke et al., FEBS Lett.464, 123-128 (1999): EBY.18ga (MATa Δhxt1-17 Δgal2 Δagt1 Δstl1 leu2-3,112 ura3-52 trp1-289 his3-Δ1 MAL2-8^(c) SUC2), EBY.VW4000 (MATa Δhxt1-17Δgal2 Δagt1 Δmph2 Δmph3 Δstl1 leu2-3, 112 ura3-52 trp1-289 his3-Δ1MAL2-8^(c) SUC2). The media were based on 1% yeast extract and 2%peptone (YP), while the minimal media were composed of 0.67% Difco yeastnitrogen base without amino acids (YNB) and comprised additives forauxotrophism requirements and different carbon sources. The yeast cellswere grown aerobically at 30° C. on an orbital shaker or on Agar plates.Cell growth was monitored by measuring the optical density at 600 nm(OD₆₀₀).

Glucose Uptake Determination

The glucose transport was measured as the uptake of D-[U—¹⁴C]-glucose(Amersham), and the kinetics parameters were determined fromEadie-Hofstee graphs. The cells were spun, washed with phosphate bufferand resuspended in phosphate buffer at a concentration of 60 mg (freshweight) per ml. Glucose uptake was determined at glucose concentrationsof between 0.2 and 100 mM, and the specific activity of the substrateranged between 0.1 and 55.5 kBq μmol⁻¹. Cells and glucose solutions werepreincubated for 5 minutes at 30° C. Glucose uptake was started bytreating the cells with radioactive glucose. After incubation for 5seconds, 10 ml of ice-cold stop buffer (0.1 M KiPO₄, pH 6.5, 500 mMglucose) were added, and the cells were filtered rapidly on glass fiberfilters (Ø=24 mm, Whatman). The filters were washed rapidly three timeswith ice-cold buffer, and the radioactivity incorporated was measured ina liquid scintillation counter. Inhibition by cytochalasin B (finalconcentration 20 μM, dissolved in ethanol) was measured in a15-second-uptake test with 50 mM or 100 mM radioactive glucose afterincubating the cells for 15 minutes in the presence of the inhibitor oronly of the solvent.

Construction of H2rg4g2 (SEQ ID No. 10) and H2rg1g2 (SEQ ID No. 12)

H2rg4g2 and H2rg1g2 are DNA constructs comprising an HXT2 promoter(promoter of the yeast glucose transport protein 2) linked functionallyto a GLUT4 gene (in SEQ ID No. 10) or GLUT1 gene (in SEQ ID No. 12) fromrats. An 0.5 kb Sa/I/EcoR1-GAL2 promoter fragment of plasmids GLUT1-pTV3and GLUT4-pTV3 (Kasahara and Kasahara, Biochem J. 315, 177-182 (1996);Kasahara and Kasahara, Biochim. Biophys. Acta 857, 146-154 (1997)) wassubstituted in each case for an 0.5 kb DNA fragment comprising the yeastHXT2 promoter from −452 by through +9 by (Genbank®: P23585).

Construction of YEp4H7-HsGLUT1 (SEQ ID No. 11) and YEp4H7-HsGLUT4 (SEQID No. 9)

YEp4H7-HsGLUT1 and YEp4H7-HsGLUT4 are plasmids in which a promoterfragment of positions −392 through −1 of the HXT7 promoter (promoter ofthe HXT7 gene) is linked functionally to a human GLUT1 gene (in SEQ IDNo. 11) or GLUT4 gene (in SEQ ID No. 9). The fragment of the promoterwas used since the complete HXT7 promoter is repressed by glucose.

A 0.4 kb Sacl/Spe1-MET25 promoter fragment from p426MET25 (Mumberg etal., Nucleic Acids Res. 22, 5767-5768 (1994)) was substituted with an0.4 kb DNA fragment comprising an HXT7 promoter fragment from positions−392 through −1, which had been amplified by means of PCR from an HXT7gene (Genbank®:P39004) as template, using the primers P426H7-1 (SEQ IDNo. 1) and P426H7-2 (SEQ ID No. 2), giving rise to plasmid YEp4H7 (SEQID No. 15). The human GLUT1 and GLUT4 ORFs (open reading frames) wereamplified over 10 cycles by means of PCR with the primer pairsHSG1-F7/T2-HSG1 (SEQ ID No. 3.4) for Glut1 and HSG4-F7/T2-HSG4 (SEQ IDNo. 5.6) for Glut4 and a human GLUT1 (EMBL:M20653) and human GLUT4 cDNA(Genbank®:M20747) as templates. The PCR products were reamplified over10 cycles using the primers T71-ORF (SEQ ID No. 7) and T2-HSG1 (SEQ IDNo. 4) or T2-HSG4 (SEQ ID No. 6). Upstream and downstream of the GLUTORF sequences, the PCR end products contain sequences which arehomologous to the HXT7 promoter region or the CYC1 termination region(iso-cytochrome c1) of plasmid YEp4H7. They were transformed into theyeast strain EBY.F4-1 together with the EcoR1-linearized YEp4H7,whereupon, following homologous recombination in yeast, thetransformation products were selected for uracil prototrophism in a 2%strength maltose medium.

Expression of GLUT1 and GLUT4 from Rats in a Hexose-Transport-DeficientYeast Strain

The yeast multicopy expression plasmids GLUT1-pTV3e and GLUT4-pTV3ecarry the glucose transporter genes GLUT1 and GLUT4 from rats under thecontrol of the galactose-inducible and glucose-repressible yeast GAL2promoter. In the two constructs, the GAL2 promoter was replaced by theglucose-inducible yeast HXT2 promoter. These vectors were transformedinto the yeast strain EBY.18ga (Δhxt), which is not capable of taking upany hexoses whatsoever and can therefore not grow on media with glucoseor other hexoses as the only carbon source. The cells were plated onto atryptophan-free synthetic medium with maltose as carbon source. Thetransformants were plated onto the same basal medium without maltose,but with different glucose concentrations (5 mM, 10 mM, 50 mM, 100 mM),using the replica-plating method. The transformants did not grow on thedifferent glucose media, not even when incubated for up to one week at30° C. This proves that the Glut1 and Glut4 glucose transporters do notsupport glucose uptake in a normal S. cerevisiae strain.

Glucose Uptake Via the Glut1 Transporter in Yeast Cells

It emerged that, following prolonged incubation of Glut1 transformantsof strain EBY.18ga on a glucose medium, colonies (termed suppressormutants or suppressor colonies hereinbelow) grew, and these wereobviously capable of taking up, and converting, glucose. The GLUT1 andGLUT4 transformants were therefore plated onto agar plates with a YNBmedium with 10 mM of glucose as the only carbon source. Followingirradiation with UV light at a sublethal dose, the cells were incubatedfor 7-14 days at 30° C. While no suppressor colonies were observed inthe case of the GLUT4 transformants, several suppressor colonies whichwere capable of growing on glucose grew on the agar plates with theGLUT1 transformants. Several of the GLUT1 suppressor mutants were grownover 15 generations in nonselective YP maltose medium. All cells whichhad lost their plasmids were no longer capable of growing on media withglucose as the carbon source. It was thus possible to demonstrate thatgrowth on glucose as the only carbon source was GLUT1-dependent. Afterthe retransformation of the original wild-type H2rg1g2 plasmid intothese cells, one of several yeast strains regains the ability of growingon glucose. This confirms that this strain comprises, in its genome, amutation which eliminates the inhibitory action on functional GLUT1expression. The mutated allele was termed fgy1-1 (which stands for“functional expression of GLUT1 in yeast”), and the strain was termedEBY.S7.

H2rg1g2 plasmids were isolated from other suppressor mutants, amplifiedin E. coli and transformed back into the originalglucose-transport-deficient yeast strain EBY.18ga (Δhxt). Several ofthese plasmids allowed growth on a synthetic medium with 10 mM glucoseas the only carbon source. Accordingly, these GLUT1 sequences comprisedmutations which converted the corresponding GLUT1 protein in the yeastinto a functional glucose transporter. For example, such mutantscomprised a substitution of valine with methionine at the position ofamino acid 69 (SEQ ID No. 13) or a substitution of alanine withmethionine at the position of amino acid 70 (SEQ ID No. 14). The mutantof SEQ ID No. 13 was found during screening of the mutants as describedabove. The mutant of SEQ ID No. 14 was obtained by in-vitro mutagenesisas shown hereinbelow. The principle of the in-vitro mutagenesis methodapplied is described by Boles and Miosga (1995) (Boles and Miosga, CurrGenet. 28, 197-198 (1995)). In a first PCR reaction, plasmidYEpH2-rGLUT1 (20 ng) as DNA template was employed together with theprimers seqhxt2 (SEQ ID No. 16) and glutmet2 (SEQ ID No. 17) (in eachcase 100 pmol) (PCR conditions: 95° C. 45 sec, 50° C. 30 sec. 72° C. 2min, 25 cycles, taq polymerase). The primer glutmet2 contains a basesequence which is modified over the normal GLUT1 gene and which leads toa substitution of alanine with methionine at the position of amino acid70 of GLUT1 from rats. The resulting PCR fragment was purified byagarose gel electrophoresis followed by gel extraction. In a second PCRreaction, the purified PCR fragment (20 ng) was used together withplasmid GLUT1-pTV3 (Kasahara and Kasahara, Biochem J. 315, 177-182(1996)) as DNA template (50 ng) and together with the primers seqhxt2and seq2gal2 (SEQ ID No. 18) (in each case 100 pmol) (PCR conditions:95° C. 45 sec, 54° C. 30 sec, 72° C. 2 min, 20 cycles, taq polymerase).Since the primer seqhxt2 only binds to the fragment of the first PCRreaction, only those DNA sequences were amplified in this second PCRreaction which lead to a substitution of alanine with methionine at theposition of amino acid 70. The resulting PCR fragment with the mutatedGLUT1 gene was purified by means of agarose gel electrophoresis followedby gel extraction, and exchanged for the wild-type GLUT1 gene in plasmidYEpH2-rGLUT1. This plasmid (SEQ ID No. 14) was transformed into theglucose-transport-deficient yeast strain EBY.18ga (Δhxt) and allowedgrowth on a synthetic medium with glucose as the only carbon source.

Strains of the yeast Saccharomyces cerevisiae, which take up glucose viathe Glut4 transporter

Strain EBY.S7 (Δhxt fgy1-1) apparently comprises a genome mutation, viz.fgy1-1, which allows Glut1 to become functional in yeast and to supportthe uptake of glucose across the plasma membrane into the cells.

Following transformation of strain EBY.S7 (Δhxt fgy1-1) with H2rg4g2,suppressor colonies were isolated which were capable of growth on mediawith glucose as the only carbon source.

Nine of these GLUT4 suppressor mutants were grown for over 15generations in nonselective YP maltose medium. All cells which had losttheir plasmids were likewise no longer capable of growing on 10 mMglucose media, which confirms that the earlier growth wasGLUT4-dependent. The H2rg4g2 plasmids were reisolated from the ninesuppressor strains, amplified in E. coli and transformed back into theoriginal glucose-transport-deficient yeast strain EBY.S7. None of theplasmids allowed growth on a synthetic medium with 10 mM glucose as theonly carbon source. This demonstrated that they comprised no “activated”mutant forms of GLUT4. Following retransformation of the originalwild-type H2rg4g2 plasmid into the nine, now plasmid-free, suppressorstrains, all of these strains regained the ability of growing onglucose, as opposed to transformants which comprised a control vectorwithout Glut4 transport protein gene. The corresponding mutations ofthis strain were termed fgy4-X (x=1-9). The mutated alleles fgy4-Xcaused functional GLUT4 expression of a GLUT4 gene expressed in thesestrains. The object of the invention was herewith achieved.

An overview of the yeast strains according to the invention which havebeen used is found in the table.

The table gives an overview of the yeast strains used in the presentinvention including the genotype, the growth conditions required forgrowing them, and the respective deposit number at the Deutsche Sammlungvon Mikroorganismen und Zellkulturen GmbH.

TABLE List of strains No. Strain Genotype Phenotype Plasmid DSM-No. 1EBY.18ga MATa Δhxt1-17Δgal2 Grows with 1% maltose — DSM 14031 Δagt1Δstl1 leu2-3,112 as C-source; auxotrophic ura3-52 trp1-289 his3-Δ1 forglucose, leucine, MAL2-8^(c) SUC2 tryptophan, histidine and uracil 2EBY.18ga MATa Δhxt1-17Δgal2 Grows with 0.2% glucose YEpH2-rGLUT1V69M DSM14026 Δagt1 Δstl1 leu2-3,112 or 1% maltose as C source; (selectionmarker TRP1) ura3-52 trp1-289 his3-Δ1 auxotrophic for leucine,MAL2-8^(c) SUC2 histidine and uracil 3 EBY.18ga MATa Δhxt1-17Δgal2 Growswith 0.2% glucose or YEpH2-rGLUT1A70M DSM 14027 Δagt1 Δstl1 leu2-3,1121% maltose as C source; (selection marker TRP1) ura3-52 trp1-289 his3-Δ1auxotrophic for leucine, MAL2-8^(c) SUC2 histidine and uracil 4 EBY.S7MATa Δhxt1-17 Δgal2 Grows with 1% maltose as — DSM 14032 Δagt1 Δstl1fgy1-1 C-source; auxotrophic for leu2-3,112 ura3-52 glucose, leucine,tryptophan, trp1-28,9 his3-Δ1 histidine and uracil MAL2-8^(c) SUC2 5EBY.S7 MATa Δhxt1-17 Δgal2 Grows with 0.2% glucose or YEp4H7-HsGLUT1 DSM14033 Δagt1 Δstl1 fgy1-1 1% maltose as C source; (selection marker URA3)leu2-3,112 ura3-52 auxotrophic for leucine, trp1-28,9 his3-Δ1 tryptophanand histidine MAL2-8^(c) SUC2 6 EBY.VW4000 MATa Δhxt1-17 Δgal2 Growswith 1% maltose as — DSM 14034 Δagt1 Δstl1 Δmph2 C-source; auxotrophicfor Δmph3 leu2-3,112 ura3-52 glucose, leucine, tryptophan, trp1-289his3-Δ1 histidine and uracil MAL2-8^(c) SUC2 7 EBY.f4-1 MATa Δhxt1-17Δgal2 Grows with 1% maltose as — DSM 14035 Δagt1 Δstl1 fgy4-1 C-source;auxotrophic for fgy4-1 leu2-3,112 ura3-52 glucose, leucine, tryptophan,trp1-289 his3-Δ1 histidine and uracil MAL2-8^(c) SUC2 8 EBY.f4-4 MATaΔhxt1-17 Δgal2 Grows with 1% maltose as — DSM 14036 Δagt1 Δstl1 fgy4-4C-source; auxotrophic for fgy4-1 leu2-3, 112 ura3-52 glucose, leucine,tryptophan, trp1-289 his3-Δ1 histidine and uracil MAL2-8^(c) SUC2 9EBY.f4-7 MATa Δhxt1-17 Δgal2 Grows with 1% maltose as — DSM 14037 Δagt1Δstl1 fgy4-7 C-source; auxotrophic for fgy4-1 leu2-3,112 ura3-52glucose, leucine, tryptophan, trp1-289 his3-Δ1 histidine and uracilMAL2-8^(c) SUC2 10 EBY.f4-1 MATa Δhxt1-17Δ gal2 Grows with 0.2% glucoseor YEp4H7-HsGLUT4 DSM 14038 Δagt1 Δstl1 fgy4-1 1% maltose as C source;(selection marker URA3) fgy4-1 leu2-3,112 ura3-52 auxotrophic forleucine, trp1-289 his3-Δ1 tryptophan and histidine MAL2-8^(c) SUC2 11EBY.f4-4 MATa Δhxt1-17 Δgal2 Grows with 0.2% glucose or YEp4H7-HsGLUT4DSM 14039 Δagt1 Δstl1 fgy4-4 1% maltose as C source; (selection markerURA3) fgy4-1 leu2-3,112 ura3-52 auxotrophic for leucine, trp1-289his3-Δ1 tryptophan and histidine MAL2-8^(c) SUC2 12 EBY.f4-7 MATaΔhxt1-17 Δgal2 Grows with 0.2% glucose or YEp4H7-HsGLUT4 DSM 14040 Δagt1Δstl1 fgy4-7 1% maltose as C source; (selection marker URA3) fgy4-1leu2-3,112 ura3-52 auxotrophic for leucine, trp1-289 his3-Δ1 tryptophanand histidine MAL2-8^(c) SUC2 Basal medium: 0.67% yeast nitrogen basewithout amino acids (Difco); pH 6.2. Supplementation of theauxotrophisms: leucine (0.44 mM), tryptophan (0.19 mM), histidine (0.25mM), uracil (0.44 mM). Maltose can be employed at a concentration ofbetween 1 and 2%; glucose between 0.2 and 2% (better growth at lowerconcentrations).

We claim:
 1. A method for generating a strain of the yeast Saccharomycescerevisiae comprising: a) providing a strain of Saccharomyces cerevisiaeyeast, b) eliminating the function of all hexose transporters of saidstrain of yeast of step a) by mutating or deleting the relevant genomicsequences, c) transforming said strain of yeast of step b) with a GLUT1gene in a yeast plasmid, d) subjecting said strain of step c) tomutagenesis wherein mutation allows the strain to grow on a medium withhexose as the only carbon source, said mutation not present in saidGLUT1 gene, e) isolating a strain capable of growing on said substratewith hexose as the only carbon source, f) selecting yeast cells fromsaid isolated strain yeast cells having no GLUT1 plasmid of step c), g)transforming said strain of step f) with a GLUT4 gene in a yeastplasmid, h) subjecting said strain of step g) to mutagenesis whereinmutation allows the strain to grow on a medium with hexose as the onlycarbon source, said mutation not present in said GLUT4 gene, i)isolating a second strain capable of growing on said substrate withhexose as the only carbon source, and j) selecting from the isolatedsecond strain yeast cells having no GLUT4 plasmid of step g).
 2. Themethod of claim 1, wherein the GLUT4 gene is a human GLUT4 gene, a mouseGLUT4 gene, or a rat GLUT4 gene.
 3. The method of claim 1, wherein aplasmid with the nucleotide sequence of SEQ ID NO: 9 or 10 is used inthe transforming step g).
 4. The method of claim 1, wherein the GLUT1gene is a human GLUT1 gene, a mouse GLUT1 gene, or a rat GLUT1 gene. 5.The method of claim 1, wherein a plasmid with the nucleotide sequence ofSEQ ID NO: 11 or 12 is used in the transforming step c).
 6. A method forgenerating a strain of the yeast Saccharomyces cerevisiae, said strainhaving eliminated therefrom function of all hexose transporters, saidelimination comprising mutating or deleting relevant genomic sequencesof said transporters, said strain comprising a GLUT4 gene and saidstrain comprising mutations in its genome that confer ability on saidstrain that allows said strain to grow on a substrate with a hexose asthe only carbon source, wherein said mutation is not present in theGLUT4 gene, comprising: a) providing a strain of the yeast Saccharomycescerevisiae which can not grow on substrates with hexoses as the onlycarbon source, said strain having eliminated therefrom function of allhexose transporters, said elimination comprising mutating or deletingrelevant genomic sequences of said transporters; b) transforming theyeast of step a) with a plasmid comprising a GLUT1 gene operably linkedto a promoter functional in yeast to form a GLUT1 strain; c) platingsaid GLUT1 strain of step b) on a medium comprising hexose as the onlycarbon source; d) subjecting said strain of step c) to mutagenesiswherein mutation allows said strain to grow on a medium comprisinghexose as the only carbon source, said mutation not present in saidGLUT1 gene; e) isolating a mutated strain; f) selecting from saidisolated mutated strain of step e) a second strain of yeast cells nothaving said plasmid comprising a GLUT1 gene; g) transforming said secondstrain with a plasmid comprising a GLUT4 gene operably linked to apromoter functional in yeast to form a GLUT4 strain; h) plating theGLUT4 strain of step g) on a medium comprising hexose as the only carbonsource; i) subjecting said strain of step h) to mutagenesis whereinmutation allows said strain to grow on a medium comprising hexose as theonly carbon source, said mutation not present in said GLUT4 gene; and j)isolating a plated strain.
 7. The method of claim 6, wherein the GLUT4gene is a human GLUT4 gene, a mouse GLUT4 gene, or a rat GLUT4 gene. 8.The method of claim 6, wherein a plasmid with the nucleotide sequence ofSEQ ID NO: 9 or 10 is used in the transforming step g).
 9. The method ofclaim 6, wherein the GLUT1 gene is a human GLUT1 gene, a mouse GLUT1gene, or a rat GLUT1 gene.
 10. The method of claim 6, wherein a plasmidwith the nucleotide sequence of SEQ ID NO: 11 or 12 is used in thetransforming step b).